Initial characterization of mice null for Lphn3, a gene implicated in ADHD and addiction.

Abstract

The LPHN3 gene has been associated with both attention deficit-hyperactivity disorder (ADHD) and addiction, suggesting that it may play a role in the etiology of these disorders. Unfortunately, almost nothing is known about the normal functions of this gene, which has hampered understanding of its potential pathogenic role. To begin to characterize such normal functions, we utilized a gene-trap embryonic stem cell line to generate mice mutant for the Lphn3 gene. We evaluated differential gene expression in whole mouse brain between mutant and wild type male littermates at postnatal day 0 using TaqMan gene expression assays. Most notably, we found changes in dopamine and serotonin receptors and transporters (Dat1, Drd4, 5Htt, 5Ht2a), changes in neurotransmitter metabolism genes (Th, Gad1), as well as changes in neural developmental genes (Nurr, Ncam). When mice were examined at 4-6 weeks of age, null mutants showed increased levels of dopamine and serotonin in the dorsal striatum. Finally, null mutant mice had a hyperactive phenotype in the open field test, independent of sex, and were more sensitive to the locomotor stimulant effects of cocaine. Considered together, these results suggest that Lphn3 plays a role in development and/or regulation of monoamine signaling. Given the central role for monoamines in ADHD and addiction, it seems likely that the influence of LPHN3 genotype on these disorders is mediated through alterations in monoamine signaling.

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Research Report
Initial characterization of mice null for Lphn3, a gene implicated
in ADHD and addiction
Deeann Wallisa,⁎, Denise S. Hillb, Ian A. Mendezc,1, Louise C. Abbottd, Richard H. Finnellb,2,
Paul J. Wellmanc, Barry Setlowe
aDepartment of Biochemistry and Biophysics, Texas A&M University, USA
bInstitute for Biosciences and Technology, Texas A&M Health Science Center, USA
cDepartment of Psychology, Texas A&M University, College Station, TX, USA
dDepartment of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA
eDepartment of Psychiatry, University of Florida College of Medicine, Gainesville, FL, USA
A R T I C L E I N F O A B S T R A C T
Article history:
Accepted 28 April 2012
Available online 7 May 2012
TheLPHN3genehasbeenassociatedwithbothattentiondeficit-hyperactivitydisorder(ADHD)
and addiction, suggesting that it may play a role in the etiology of these disorders.
Unfortunately, almost nothing is known about the normal functions of this gene, which has
hampered understanding of its potential pathogenic role. To begin to characterize such
normal functions, we utilized a gene-trap embryonic stem cell line to generate mice mutant
for the Lphn3 gene. We evaluated differential gene expression in whole mouse brain between
mutant and wild type male littermates at postnatal day 0 using TaqMan gene expression
assays. Most notably, we found changes in dopamine and serotonin receptors and
transporters (Dat1, Drd4, 5Htt, 5Ht2a), changes in neurotransmitter metabolism genes (Th,
Gad1), as well as changes in neural developmental genes (Nurr, Ncam). When mice were
examined at 4–6 weeks of age, null mutants showed increased levels of dopamine and
serotonininthedorsalstriatum. Finally,null mutantmicehad a hyperactivephenotype in the
openfieldtest,independentofsex,andweremoresensitivetothelocomotorstimulanteffects
of cocaine. Considered together, these results suggest that Lphn3 plays a role in development
and/or regulation of monoamine signaling. Given the central role for monoamines in ADHD
and addiction, it seems likely that the influence of LPHN3 genotype on these disorders is
mediated through alterations in monoamine signaling.
© 2012 Elsevier B.V. All rights reserved.
Keywords:
Lphn3
Latrophilin
Addiction
Attention deficit-hyperactivity
disorder
1. Introduction
Addiction and ADHD are highly prevalent and costly to our
society, but there is a current lack of reliable diagnostic or
pharmacogenetic biomarkers for either disorder, and ther-
apeutic modalities need significant improvement. Recently
LPHN3 has been identified by linkage and association studies
performed in separate cohorts by separate research groups as
B R A I N R E S E A R C H 1 4 6 3 ( 2 0 1 2 ) 8 5 – 9 2
⁎ Corresponding author at: Department of Biochemistry and Biophysics, Interdisciplinary Life Sciences Building Rm 2146B, Texas A&M
University, College Station, TX 77843‐3474, USA. Fax: +1 979 862 7638.
E-mail address: dwallis@tamu.edu (D. Wallis).
1Current address: Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, USA.
2Current address: Department of Nutritional Sciences, The University of Texas at Austin, Austin, TX, USA.
0006-8993/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2012.04.053
Available online at www.sciencedirect.com
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a prime candidate for both addictive phenotypes and ADHD.
Vulnerability to addiction is a complex trait with strong genetic
influences that are largely shared by abusers of different
addictive substances. Initially, alcohol abuse was linked to
Chromosome 4 less than 300 kb 5′ of the LPHN3 gene (Long et
al., 1998). Another independent study identified marker
D4S244 (also on Chromosome 4) linked to alcohol depen-
dence (Reich et al., 1998; Uhl, 2004a,b). Additional evidence
was supplied by a third study that utilized whole genome
association analysis to identify SNPs with significant allele
frequency differences between abusers of illegal substances
and control populations of both European-American and
African-American ethnicities (Liu et al., 2006). This research
group replicated these previously-identified LPHN3 SNPs as well
as additional LPHN3 SNPs in several different populations
including polysubstance abusers (Bergen et al., 1999), alcohol
dependent samples (Johnson et al., 2006; Liu et al., 2006), and
methamphetamine dependent samples from Japan and Taiwan
(Uhl et al., 2008a,b). Finally, several groups have performed
genome wide linkage and association studies for ADHD
(reviewed by Franke et al., 2009), and LPHN3 has been
recently associated with attention deficit-hyperactivity dis-
order (ADHD) (Arcos-Burgos et al., 2010). This group has also
found that ADHD and Chromosome 4 loci (in the LPHN3 area)
are linked to addiction (Jain et al., 2007).
LPHN3(latrophilin3)isamemberofthelatrophilinsubfamily
of secretin G protein coupled receptors (GPCR) (Matsushita et al.,
1999). LPHN1 and LPHN2 are receptors for latrotoxin (black
widow spider venom) (Ichtchenko et al., 1999), which interacts
with neuronal GPCRs to stimulate exocytosis of neurotransmit-
ters (Krasnoperov et al., 1997;Lelianova etal., 1997). Structurally,
LPHNs appear to be chimeras between cell surface receptors
and intracellular signaling molecules (Krasnoperov et al., 2002);
however, the endogenous ligands for all three LPHNs are
unknown, and all functional studies of LPHNs to date have
relied on the interaction of latrotoxin with LPHN1 and LPHN2.
Currently, no functional polymorphisms within LPHN3 have
beenidentifiedthatmightexplaindiseasesusceptibility.Arecent
mutational analysis of the entire coding region of LPHN3 in a
cohortof139ADHDsubjectsand52controlswasconductedinan
attempt to identify susceptibility or protective haplotype alleles.
Althoughsomenovelpolymorphismswereidentified,nonewere
associated with obviously significant coding region changes, or
canonical splice site alterations (Domené et al., 2011). This
suggests that non-coding variations determining the quantity
and/orqualityofLPHN3isoformsarelikelycontributorstoADHD,
but the mechanisms by which LPHN3 might influence ADHD
susceptibility (or, indeed, any LPHN3 mechanisms at all), are
essentially unknown. To address these significant data gaps in
ourknowledgeofthefunctionsofthispotentiallyveryimportant
gene, we generated a Lphn3 null mutant mouse, in the hope that
characterization of this mutant would enhance our understand-
ing of the pathophysiology of both ADHD and addiction. Here we
report preliminary cellular, neurochemical, and behavioral char-
acteristics of these mice.
2. Results
2.1. Lphn3 mutant mouse generation
We utilized gene trap clone FHCRC-GT-S17-5H1 generated with
the ROSAFARY vector (Chen et al., 2004) to generate mutant
mice; 129S4/SvJae and C57 mix. A schematic representation of
this clone (Fig. 1A) depicts both the transcript structure and the
location of the trap based on RACE sequence. We generated
genomic sequence confirming an insertion which is predicted
to interrupt the mucin stalk domain in the middle of the gene.
Such a mutation is predicted to obliterate any intracellular
signaling from Lphn3, if not all functions. We injected the clone
into blastocysts and produced germline mutations. The nulls
were viable and appeared physically indistinguishable from
Fig. 1 – Lphn3 gene-trap and RT-PCR. A: Screen shot of mouse Lphn3 genomic region and NCBI transcript Lphn3 showing exon
and intron structure. Below the transcript line is the raw 3′ RACE sequence alignment from the gene-trapped ES cell clone used
to generate the Lphn3 deficient mice. B: RT-PCR verifying knockout of Lphn3 mRNA in null mice.
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wild type littermates, with no evident mortality or morbidity
(up to 5 months). Body weight data from a subset of the mice
(obtainedat1and2,andinsomecases3 monthsofagefromthe
samemice: ns=14 (10) null,20(13)heterozygous, and 14 (5) wild
type; numbers in parentheses indicate ns for which body
weights were recorded at 3 months) showed no differences
between genotypes at any age (one-factor ANOVAs, Fs<1.42,
ps>.25). Normal sex ratios and Mendelian relative genotype
ratios of 1:2:1 were observed after heterozygous crossings
(N=201 pups). Disruption of the Lphn3 transcript was verified
with RT-PCR probes generated to the Lphn3 mRNA flanking the
gene-trap insertion site (Fig. 1B).
2.2. Gene expression analysis
Because both ADHD and addiction have been linked to altered
monoaminergic signaling, we examined the expression of genes
involved in differentiation, survival, and function of dopaminer-
gic and serotonergic neurons and neurotransmitter function in
experimentally naïve wild type and Lphn3 null mice (n=6 mice/
group at P0). ADHD candidate genes were included as well, as
were loci previously suspected to interact with Lphn3. Specifi-
cally, we looked for changes in expression levels of the genes for
Nurr1, Ncam, TH, DAT, DA receptors (Drd2, 4, and 5), serotonin
transporter (5-Htt), serotonin receptor 2A, and Gad67 by Q-RT-
PCR. Dopamine transporter and receptors are key for both
dopaminergic function and as ADHD candidate genes. Dat1
and Drd4 are the most highly replicated ADHD candidate genes.
Drd2 has been implicated in both alcoholism and ADHD. Drd5 is
alsoanADHDcandidategene.ThisrequiredforsynthesisofDA.
5-Htt and 5-Ht2a are also ADHD candidate genes and regulate
the serotonin system. GAD67 catalyzes the conversion of
glutamic acid to gamma-aminobutyric acid (GABA), the major
inhibitory neurotransmitter in the vertebral central nervous
system. Ncam has been implicated as having a role in cell–cell
adhesion, neurite outgrowth, synaptic plasticity, and learning
and memory. Further, single-nucleotide polymorphisms (SNPs)
harboredintheLPHN3geneinteractwithSNPsspanningthe11q
region that contains DRD2 and NCAM1 genes, to double the risk
of developing ADHD (Jain et al., 2011). Nurr1 plays a key role in
the maintenance of the dopaminergic system of the brain and is
essential for generation of midbrain dopamine cells during
embryonic development.
We observed relative statistically significant differential
expression between wild type and null mutants based on
Lphn3 genotype for: 5-Htt, 5-Ht2a, Dat1, Drd4, Ncam, Nurr1,
and TH (Fig. 2 and Table 1) (all p<0.01). Verification of RT and
Q-PCR results were obtained after repeating the RT and gene
expression assays a second time on the same RNA samples.
2.3.Neurochemical analysis
In a separate experiment involving an experimentally naive
cohort of mice (n=6 null, 13 heterozygotes, and 10 wild type),
we evaluated levels of dopamine and serotonin in the dorsal
striatum at 4–6 weeks of age. One-factor ANOVA revealed
main effects of genotype for both dopamine (F(2, 26)=3.36,
p=.05) and serotonin (F(2, 25)=5.05, p<.05). Post-hoc compar-
isons (Bonferroni contrasts) showed that Lphn3 null mice had
significantly higher DA and 5-HT levels than WT mice (p<.05),
but no other differences between groups were significant
(Fig. 3).
2.4.Locomotor activity
We evaluated male and female mice (n=15 null, 19 hetero-
zygous, and 14 wild type) for activity levels in an open field
arena at 4, 8, and 12 weeks of age. A multi-factor ANOVA
(genotype×sex×age) was used to compare data from four
activity measures (horizontal activity, vertical activity, stereo-
typy activity, and center time). On the horizontal activity
measure, there was a main effect of genotype (F(2, 42)=14.92,
p<.01), but no main effects or interactions involving age or sex
(ps>.20) (Fig. 4A). Post-hoc comparisons revealed that null
mice showed significantly more horizontal activity than both
heterozygous and wild type mice (ps<.01), but that hetero-
zygous and wild type mice did not differ. On the vertical
activity measure, there was a main effect of age (F(2, 84)=8.74,
p<.01), but no other main effects or interactions involving sex
or genotype (data not shown, ps>.11). On the stereotypy
measure, there was also a main effect of age (F(2, 84)=4.36,
p<.05), as well as a main effect of genotype (F(2, 42)=7.01,
p<.01), but no other significant effects (ps>.17). Post-hoc
comparisons between genotype revealed that, as with the
horizontal activity measure, null mice showed significantly
Fig. 2 – Differential gene expression in P0 male Lphn3 null
mutant mice. Asterisks indicate genes for which there was a
statistically significant increase in expression compared to
expression in wild type mice.
Table 1 – Q-RT-PCR results comparing fold changes in
expression of specific genes between Lphn3 wildtype and
null mice and their statistical significance levels.
GeneFold diff t test
Mm5-Htt
MmDrd2
MmDrd5
Mm5-Ht2a
MmDat1
MmDrd4
MmGad
MmNcam
MmNurr
MmTh
2.192468
0.957235
1.374059
2.544708
2.749919
2.921976
3.150445
2.937998
2.937998
3.366177
0.000725
0.43198
0.158794
1.94E-05
4.79E-05
6.48E-06
0.003392
0.001139
0.001139
0.001188
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more stereotypy activity than both heterozygous and wild type
mice(ps<.05),but that heterozygousand wildtypemice did not
differ (data not shown). On the time in center measure, there
wasasignificantinteractionbetweenageand sex(F(2,84)=3.64,
p<.05) such that males spent more time in the center of the
open field than females at 8 and 12 weeks of age. The main
effect of genotype approached statistical significance (p=.07,
with null mice showing greater time in center than wild type),
but there were no other significant main effects or interactions
(data not shown, ps>.16).
2.5. Locomotor response to cocaine
To determine whether Lphn3 null mice were differentially
sensitive to the locomotor stimulant properties of psychosti-
mulants compared to wild type littermates, we evaluated the
effects of acute administration of two doses of cocaine using a
within-subjects design (n=14 null and 13 wild type mice that
had been previously tested for locomotor activity). Analyses
were focused on the horizontal activity measure, as the
effects of genotype were most robust on this measure. As in
the assessment of locomotor activity, null mice displayed
significantly greater horizontal activity than wild type mice
during the pre-drug habituation period (two-factor ANOVA
(genotype×time bin), main effect of genotype, F(1,23)=12.32,
p<.01, data not shown). To account for the elevated baseline
activity levels in null mutant mice, we subtracted baseline
activity counts (in the final time bin of the habituation period)
from the counts in each time bin following drug administra-
tion. A three-factor ANOVA (drug×genotype×time bin) con-
ducted on these normalized data revealed a main effect of
drug (F(2, 46)=6.81, p<.01), such that cocaine administration
elevated locomotor activity, and bin (F(11, 253)=11.79, p<.01),
such that across groups, activity decreased over the course of
the test session. Importantly, there was a 3-way interaction
between drug, genotype, and bin (F(22, 506)=1.64, p<.05), such
that Lphn3 null mice showed a greater increase in activity in
response to the 20 mg/kg dose of cocaine than their wild type
cohorts (Fig. 4B). There were no significant main effects or
interactions involving sex.
3. Discussion
The LPHN3 gene has been linked to both ADHD and addictive
disorders, but the basic functions of the protein product of
this gene are almost entirely unknown. The experiments
presented here represent an initial attempt at characteriza-
tion of this gene's function using mice with a null mutation in
the Lphn3 gene (hence, we are more literally characterizing the
functional consequences of deletion of Lphn3 in these mice).
The resulting alterations in gene expression, neurochemistry,
and behavior suggest that Lphn3 plays a role in development
and/or regulation of monoaminergic signaling. This suggests
in turn that the link between LPHN3 and ADHD/addiction in
humans may be mediated by altered monoamine signaling.
Genes in the Lphn family have been implicated in neuro-
transmitter release due to their affinity for latrotoxin (Sudhof,
2001), but unlike the other members of this family (Lphn1 and 2),
Lphn3doesnotbindlatrotoxin.Todate,alteredexpressionlevels
ofLphn3havebeenseenafterbrainischemia(BinSunetal.,2002)
and during development of the adrenal gland (Xing et al., 2009).
Lphn3 has also been implicated as a tumor suppressor based on
Fig. 3 – Dopamine and serotonin levels (as determined by
HPLC)inwildtype,heterozygous,andLphn3nullmutantmice.
Null mutantshad significantlygreater levels ofbothdopamine
and serotonin compared to wild type, but heterozygotes did
not differ significantly from either group.
A
Lphn3 mutant mouse
locomotor activity
48 12
0
500
1000
1500
2000
2500
wild-type
heterozygous
null
Age (weeks)
Horizontal Activity (arbitrary units)
B
Lphn3 mutant
locomotor activity
in response to cocaine
024
bin (5 min)
68 10 12
0
1000
2000
3000
wild type saline
null saline
wild type 5 mg/kg cocaine
null 5 mg/kg cocaine
wild type 20 mg/kg cocaine
null 20 mg/kg cocaine
Horizontal Activity (arbitrary units)
Fig. 4 – Open field activity in Lphn3 mice. A: Locomotor
activity in wild type, heterozygous, and Lphn3 null mutant
mice at 4, 8, and 12 weeks of age. Lphn3 null mutants
displayed significantly greater activity than wild type at all
ages, but heterozygous and wild type mice did not differ.
B: Locomotor activity in wild type and Lphn3 null mutant
male and female mice in response to acute administration of
0, 5, and 20 mg/kg cocaine. Lphn3 mutant mice showed a
significantly enhanced locomotor response to 20 mg/kg
cocaine, compared to wild type.
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identified copy number losses and various protein-altering
missense mutations in human cancers, particularly lung cancer
(Kan et al., 2010). The spatial and temporal expression of LPHN3
supportsitsroleinthepathogenesisofADHD.Inhumans,LPHN3
is temporally and spatially dynamic such that its expression
decreases as the brain matures, and ultimately remains detect-
ableprimarilyinregionsofthebrainindependentlyimplicatedin
ADHD pathogenesis (Arcos-Burgos et al., 2010). We have also
identified Lphn3 expression in the developing embryonic mouse
brain, as well as in the adult mouse hippocampus and thalamus
(data not shown). It is also of note that the dosage of the LPHN3
susceptibility haplotype in humans varies inversely with the
ratio of NAA/Cr in the right medial and lateral thalamus (Arcos-
Burgos et al., 2010). Further, The NAA/Cr ratio increases mono-
tonically in the right medial and lateral thalamus in relation to
the number of copies of the protective haplotype. The ratio of
NAA/Cr is a measure of the neuronal number thought to be
abnormal in ADHD. Finally, the same LPHN3 variant associated
with susceptibility to ADHD is also associated with response to
stimulant medication (Arcos-Burgos et al., 2010). Unfortunately,
the published literature does not indicate any mechanism of
action for LPHN3 nor a potential role in behavior.
The alterations observed in the Lphn3 null mutant mice are
consistentwitha role for thisgene inmonoaminergic signaling.
Newborn null mutant mice showed elevated whole brain
expression of several genes involved in dopamine (Dat1, Drd4,
TH) and serotonin (5-Htt, 5-Ht2a) signaling, as well as neurode-
velopmentofthesesystems(Ncam,Nurr).Interestingly,manyof
these genes have also been linked independently to ADHD and/
or addiction, (Anastasio et al., 2011; Bobb et al., 2005; Eells, 2003;
Matzel et al., 2008; Wallis et al., 2008; Werme et al., 2003)
suggesting a further link between Lphn3 and these conditions.
In addition to changes in monoaminergic gene expression,
alterations in monoamine neurochemistry were also observed
in null mutant mice. Specifically, at 4–6 weeks of age, null
mutants showed elevated levels of both dopamine and
serotonin compared to wild type, while heterozygotes showed
an intermediate phenotype. This elevation in tissue mono-
amine content would seem to run contrary to the elevations in
expression of genes for both dopamine and serotonin transpor-
ters also observed in the null mutants, although it is possible
that the upregulation in monoamine transporter genes repre-
sents a compensatory response to elevated monoamine synth-
esis (which would be consistent with the upregulation in Th
expression). Given the different timepoints and brain regions
sampled, more work will be needed to determine the spatial
and temporal specificity of these effects and their relationship
to one another. The fact that 9 of 10 of these genes assessed by
Q‐RT‐PCR were overexpressed, coupled with the increases in
both of the neurotransmitters DA and 5-Ht, suggests that there
are indeed problems with neurotransmitter homeostasis and
neuronal differentiation, development, and/or function as we
predicted in these mutant mice. This suggests that Lphn3
deficiency results in disruptions of homeostasis for these genes
and neurotransmitters, although the mechanisms of this dis-
ruption are as of yet unknown.
At the behavioral level, null mutant mice showed a robust
elevation in horizontal locomotor activity relative to both
heterozygous and wild type mice, which was present across
multiple timepoints beginning at 4 weeks of age (a period
corresponding to early adolescence in the mouse). This same
pattern of behavior was also evident in measures of vertical
activity (rearing) and stereotypy (short, repeated movements),
although the effect in vertical activity did not reach statistical
significance. Null mutant mice also showed a trend toward
more time spent in the center of the open field; such an
increase in center time could be interpreted as decreased
anxiety, but it is difficult to separate such effects from the
hyperactivity also present in these mice.
There was no statistical evidence for an age-dependent
decreaseinhyperactivityinnullmutantmice(i.e.,nointeraction
between age and genotype); however, visual inspection of the
data (Fig. 4A) suggested that hyperactivity in the null mutant
mice may have begun to decrease by the third test period
(12 weeks of age), and exploratory post-hoc analyses revealed
that the difference between null mutant and wild type mice no
longer reached statistical significance at this time point. It is not
clear whether this potential decrement represents a true age-
dependent decrease in the hyperactive phenotype or a habitua-
tion to the locomotor testing environment. Such an attenuation
would be consistent with the age-dependent decrease in ADHD
symptoms observed in some cohorts (Biederman et al., 1996; Hill
and Schoener, 1996). However, it is important to note that at
4 months of age (when cocaine testing took place), null mice still
displayed hyperactivity during the baseline, pre-drug period,
indicating that the hyperactive phenotype is still present well
into adulthood.
The hyperactivity displayed by the Lphn3 null mutant mice
is consistent with the elevated striatal monoamine (particu-
larly dopamine) content observed at 4–6 weeks of age. Indeed,
elevated striatal dopamine is associated with locomotor hyper-
activity in other mutant mouse lines, including the dopamine
transporter knockout mice (Gainetdinov et al., 1999). Notably,
these Dat1 mice have been proposed to model some features of
ADHD(Davids et al., 2003;Mill, 2007; Russell,2011; Russell, 2007;
vanderKooij and Glennon,2007),suggesting thattheLphn3null
mutant mouse may have some utility in this regard. In contrast
to these other models, however, (in which stimulant drugs
reverse the hyperactive phenotype), Lphn3 null mutant mice
showed an enhanced locomotor response to stimulant drug
(cocaine) administration compared to wild type controls, indi-
cating that the relationship between monoaminergic dysfunc-
tion, hyperactivity, and response to stimulant drugs is complex.
Since initial submission of this manuscript and during the
review process, some very important and relevant studies on
Lphn3 have been published. A zebrafish model has been
developed (Lange et al., 2012). Loss of lphn3.1 function causes
a reduction and misplacement of dopamine-positive neurons
in the ventral diencephalon and a hyperactive/impulsive
motor phenotype. The behavioral phenotype can be rescued
by the ADHD treatment drugs methylphenidate and atomox-
etine. Interestingly, the zebrafish model was generated using
morpholinos that caused only transient downregulation of
lphn3.1. In addition, as zebrafish contain two orthologs of
LPHN3 (due to genome duplication in teleost fish), morpho-
lino knockdown oflphn3.1 (and not lphn3.2) may cause only a
hypomorphic reduction in Lphn3 protein, an important
consideration for ADHD etiology. While the hyperactivity
observed in the zebrafish corresponds to the hyperactivity we
observe in the mouse, our data showing alterations of DA and
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5-HT in the dorsal striatum at 4–6 weeks of age in the mouse
is inconsistent with data indicating no changes in DA in
zebrafish larvae brain. This discrepancy may be due to
differences in developmental time points between the two
species, the fact that we sampled specifically from the dorsal
striatum as opposed to global brain tissues, the transient
nature of morpholino knockdown, or the fact that zebrafish
still have lphn3.2 for potential compensation of the pheno-
type. Asecondrecentandverysignificantdevelopmentwasthe
identification of FLRT proteins as endogenous LPHN ligands
(O'Sullivan et al., 2012). This groups reports that the FLRT3 and
LPHN3 ectodomains interact with high affinity in trans and
conclude that LPHN3 and its ligand FLRT3 play an important
role in glutamatergic synapse development. They also show
that hippocampal cultures transfected with shRNA against
Lphn3 have a decreased number of excitatory synapses.
In summary, the data gathered thus far suggest that Lphn3
null mutant mice display evidence of profound disruption at
multiple levels of monoamine signaling. Future research will
expand upon these data to further characterize this pheno-
type at both the molecular (neuroanatomy, gene and protein
expression) and behavioral (different drugs of abuse, atten-
tion, impulsivity) levels. Given the established links between
LPHN3 and ADHD/addiction, such characterization may lead
to enhanced understanding of the pathophysiology of these
disorders, and potentially yield novel therapeutic targets or
biomarkers for diagnostics or prediction of treatment success.
4. Experimental procedures
4.1.Clone expansion, confirmation and microinjection
Gene-trapped murine embryonic stem (ES) cells were thawed
and expanded. Sequenceverificationofthe insertion site by the
meansofinversegenomic PCR(IPCR)confirmedthecorrectgene
targeting event within Lphn3. Once the ES cells were expanded
and ready for injection, they were trypsinized and re-suspended
in 5 ml of injection buffer (DMEM, 10% FCS, 2 mM glutamine,
20 mM HEPES) and kept on ice. ES cells were injected into C57BL/
6 strain blastocysts. Twenty to thirty blastocysts were injected
per sitting. Injected blastocysts were transferred to the uterine
horn of 2.5 dpc pseudopregnant CD-1 recipients. Pups were born
17 days later and chimeric mice were identified 7–10 days later
based on their coat color. High percentage male chimeras were
mated to C57BL/6–albino females to test for germline transmis-
sion through gene specific PCR genotyping.
4.2. Mice
All animals were genotyped from genomic DNA isolated from
tails collected at weaning using Extract-N-Amp (Sigma Aldrich,
St. Louis, MO). All mice tested for molecular and behavioral
assaysweresiblingcohorts,andwerehousedingroupsof5mice
per cage in an animal room at 20–22 °C, under a 12-hour light/
dark cycle (on at 7:00 h) with ad libitum access to food and water.
All procedures were approved by the Texas A&M University
Institutional Animal Care and Use Committee and carried out in
accordance with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals (National Institutes of
Health Publication No. 85–23, revised 1996). Mice were eutha-
nized by CO2asphyxiation and cervical dislocation prior to
tissue harvest.
4.3.Q-RT-PCR
Whole brains were harvested from P0 pups and stored in
RNAlater (Ambion, Austin, TX). RNA was extracted from 6
male null and 6 male wild type mice using Qiagen RNEasy kits
and quantified. RNA was reverse transcribed with SuperScript III
(Invitrogen) and Q-PCR was performed using gene specific
TaqMan assays. Gapdh expression was used for normalization.
Folddifferentialexpressionbetweenmutantsandwildtypeswas
calculated using the delta–delta Ct method as recommended by
the manufacturer (Applied Biosystems, Carlsbad, CA).
4.4. HPLC
Whole brain tissue was harvested from 4 to 6 week old male
mice (n=6 null, 13 heterozygotes, and 10 wild type). Brains
were extracted and snap frozen, slices were made and tissue
punches through the striatum were collected. Tissue con-
centrations of dopamine and serotonin were determined
using high pressure liquid chromatography (Kramer et al.,
2007; Shah et al., 2005; Sved, 1989). Just prior to assay, the
tissue samples were weighed and sonicated in perchloric
acid containing dihydrobenzylamine (DHBA; an internal
standard, 100 ng/ml: ESA, Chelmsford, MA). The amines
were passed through a low volume nylon 0.2-μm filter
(Model 8110: Fisher Scientific, Houston, TX) and the resulting
supernatant was injected onto a reversed phase C-18 column
(Shiseido, 5 cm, Model # A3RE01176: ESA: Chelmsford, MA).
The sample amines were eluted using a filtered and degassed
mobile phase (Fast Dopamine: ESA, Chelmsford, MA) and
then quantified by electrochemistry (Coulochem II: ESA
Chelmsford, MA) using a microdialysis cell (Model 5014B:
ESA: Chelmsford, MA). Sample dopamine and serotonin peak
heights were compared with external standard peak heights
(Sigma Chemical, St. Louis, MO) that were processed in a
manner similar to that of the samples. Sample values were
expressed as amine concentrations (pg) per mg wet weight of
sample tissue.
4.5. Locomotor activity
Fifteen null (6 female; 9 male), 19 heterozygous (9 female;
10 male), and 14 wild type (9 female; 5 male) mice were
used to assess locomotor activity. Locomotor activity was
assessed during the light cycle in 8 standard activity cham-
bers (40×40×30 cm, Accuscan Instruments: Columbus, OH)
under dim light and white noise conditions. Mice were placed
into the chambers, and activity data were collected and saved
to a computer file via an array of photobeams that crossed the
open field at two heights. Activity was measured over 30 min
in six bins of 5 min each. Eachmousewastestedat3timepoints
in an identical manner (4, 8, and 12 weeks of age, which
corresponds to early adolescence, late adolescence, and adult-
hood). Four measures of activity were used to assess behavior in
the open field: horizontal activity (total number of breaks of the
lower set of photobeams), vertical activity (total number of
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breaks of the upper set of photobeams—i.e., rears), stereotypy
activity (number of repeated breaks of the same photobeam or
set of photobeams) and center time (amount of time spent more
than 1 cm from the wall of the open field).
4.6. Cocaine administration
Fourteen null (5 female; 9 male) and 13 wild type (8 female;
5 male) mice (ages 4–5 months) used in the locomotor activity
tests were also used to assess the effects of acute cocaine
administration on locomotor activity. Following a 30-minute
habituation period (six 5-minute bins) in the activity cham-
bers, mice were given acute i.p. injections of one of three
doses of (−) cocaine HCl (NIDA Drug Supply Program, 0, 5, and
20 mg/kg in physiological saline vehicle, 10 ml/kg) immedi-
ately prior to a 60-minute locomotor activity test session
(twelve 5-minute bins). Each mouse received each dose of
cocaine using a within-subjects design, and the order of doses
was randomized by sex and genotype. At least 48 h elapsed
between successive injections. The doses of cocaine were
calculated as the weight of the salt and were chosen based on
their ability to stimulate locomotor activity in mice (Dudek et al.,
1991; Phillips et al., 1998).
4.7. Statistical analysis
Gene expression and neurochemical data were analyzed via
independent samples t-tests. Data from the locomotor activ-
ity tests were analyzed using multi-factor repeated measures
ANOVA (sex×genotype×time point). Data from the cocaine
administration test were also analyzed using multi-factor
repeated measures ANOVA (sex×genotype×drug dose×time
bin). Bonferroni post-hoc comparisons were used when appro-
priate (in the presence of main effects or interactions in the
ANOVAs). In all cases, p≤.05 were considered significant.
Acknowledgments
The Texas A&M Institute for Genomic Medicine generated the
Lphn3 nullmiceand providedfunding forthisproject. We thank
Alex Ivanov, Colin Vokes, and Rebecca Hofford for technical
assistance.
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